Use of parthenolide derivatives as antileukemic and cytotoxic agents

ABSTRACT

The present invention provides compounds of the formula (I) 
     
       
         
         
             
             
         
       
         
         
           
             wherein: 
             X 1 , X 2  and X 3  are heteroatoms; 
             R 4 , R 5 , R 6 , R 7 , R 8 , R 9  and R 10  are independently selected from H, halo, —OH, —NO 2 , —CN and optionally substituted aliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and 
             Z is optionally substituted C 1-8  straight-chained or branched aliphatic, optionally containing 1 or more double or triple bonds, wherein one or more carbons are optionally replaced by R* wherein R* is optionally substituted cycloalkyl, heterocycloalkyl, aryl or heteroaryl; an amino acid residue, H, —CN, —C(O)—, —C(O)C(O)—, —C(O)NR 1 —, —C(O)NR 1 NR 2 —, —C(O)O—, —OC(O)—, —NR 1 CO 2 —, —O—, —NR 1 C(O)NR 2 —, —OC(O)NR 1 —, —NR 1 NR 2 —, —NR 1 C(O)—, —S—, —SO—, —SO 2 —, —NR 1 —, —SO 2 NR 1 —, —NR 1 R 2 , or —NR 1 SO 2 —, wherein R 1  and R 2  are independently selected from H and optionally substituted aliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; or where R* is NR 1 R 2 , R 1  and R 2  optionally together with the nitrogen atom form an optionally substituted 5-12 membered ring, said ring optionally comprising 1 or more heteroatoms or a group selected from —CO—, —SO—, —SO 2 — and —PO—; or 
             a pharmaceutically acceptable salt, ester or prodrug thereof.

This is a divisional application of U.S. patent application Ser. No.13/372,178, filed Feb. 13, 2012, now U.S. Pat. No. 8,470,875, issuedJun. 25, 2013, which is a divisional of U.S. patent application Ser. No.12/693,161, filed Jan. 25, 2010, now U.S. Pat. No. 8,124,652, which is adivisional application of U.S. patent application Ser. No. 11/031,315filed Jan. 7, 2005, now U.S. Pat. No. 7,678,904, which is acontinuation-in-part of U.S. patent application Ser. No. 10/888,274filed Jul. 9, 2004, now U.S. Pat. No. 7,312,242, which claims thebenefit of priority to provisional application No. 60/486,171 filed Jul.11, 2003.

FIELD OF THE INVENTION

The present invention relates to methods for the structural modificationof the sesquiterpene lactone, parthenolide, and the use of theseparthenolide derivatives in the treatment of carcinoma. Morespecifically, the invention relates to the methods to prepare structuralanalogs of the parent compound, parthenolide, in order to obtain new,pharmacologically active chemical entities with improved watersolubility characteristics, and to use them in the treatment ofleukemias and other parental and multi-drug resistant cancers

BACKGROUND OF THE INVENTION

Sesquiterpene lactones are a group of secondary plant metabolitesconsisting of a 15-carbon structure containing anα-methylene-γ-butyrolactone moiety and other additional functionalgroups. Over the last two to three decades, these terpenoids havereceived considerable attention due to the broad spectrum of theirbiological activities, to the plants which produce them, and mostimportantly, because of their pharmacological effects in humans. About4,000 of these terpenoids have been isolated and identified, most ofthem in Asteraceae (Compositae, sunflower family) (Schmidt, Curr. Org.Chem. 1999, 3, 577-608). Some of these plants have been used forcenturies in indigenous medical practices in various cultures worldwide.

Parthenolide (1) is a Germacrane sesquiterpene lactone with a uniquestructure. It has been isolated from several different species inAsteraceae (Compositae) family, feverfew (Tanacetum parthenium) beingone of them.

Feverfew has been used to reduce fever and pain and in the treatment ofmigraine and rheumatoid arthritis (Heptinstall et al., ACS SymposiumSeries (1998), 691 (Phytomedicines of Europe), 158-175). The activecomponent is parthenolide (1). Recently, it has been revealed thatparthenolide (1) can induce tumor apoptosis by the inhibition of NF-κBactivities (Cory et al., Anticancer Research 2002, 22, 3805-9; Cory etal., Anticancer Research 2001, 21, 3807-11; Gelfanov et al., Blood,2000, 98, 2508-17; Kang et al, Brit. J. Pharmacol. 2002, 135, 1235-44;Song et al., J. Asian. Nat. Prod. Res. 2001, 3, 285-91).

Parthenolide (1) is a lipophilic, neutral lactone with low polarity, andhas a low water-solubility, limiting its development as a therapeuticagent. Thus, a need exists for the development of soluble parthenolidederivatives that retain their anti-cancer activity.

SUMMARY OF THE INVENTION

In accordance with the present invention, a novel class of compoundswith antileukemic activity is presented. Accordingly, the presentinvention provides compounds of formula (I):

-   -   wherein:    -   X₁, X₂ and X₃ are heteroatoms;    -   R₄, R₅, R₆, R₇, R₈, R₉ and R₁₀ are independently selected from        H, halo, —OH, —NO₂, —CN and optionally substituted aliphatic,        cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and        Z is optionally substituted C₁₋₈ straight-chained or branched        aliphatic, optionally containing 1 or more double or triple        bonds, wherein one or more carbons are optionally replaced by R*        wherein R* is optionally substituted cycloalkyl,        heterocycloalkyl, aryl or heteroaryl; an amino acid residue, H,        —CN, —C(O)—, —C(O)C(O)—, —C(O)NR¹—, —C(O)NR¹NR²—, —C(O)O—,        —OC(O)—, —NR¹CO₂—, —O—, —NR¹C(O)NR²—, —OC(O)NR¹—, —NR¹NR²—,        —NR¹C(O)—, —S—, —SO—, —SO₂—, —SO₂NR¹—, —NR¹R², or —NR¹SO₂—,        wherein R¹ and R² are independently selected from H and        optionally substituted aliphatic, cycloalkyl, heterocycloalkyl,        aryl or heteroaryl; or where R*is NR¹R², R¹ and R² optionally        together with the nitrogen atom form an optionally substituted        5-12 membered ring, said ring optionally comprising 1 or more        heteroatoms and/or a group selected from —CO—, —SO—, —SO₂— and        —PO—; or    -   a pharmaceutically acceptable salt, ester or prodrug thereof.

The invention also provides a pharmaceutical composition comprising aneffective amount of a compound of formula (I), or a pharmaceuticallyacceptable salt, ester or prodrug thereof, in combination with apharmaceutically acceptable diluent or carrier.

The invention also provides a method of inhibiting cancer cell growthand metastasis of cancer cells, comprising administering to a mammalafflicted with cancer, an amount of a compound of formula (I), effectiveto inhibit the growth of said cancer cells.

The invention also provides a method comprising inhibiting cancer cellgrowth by contacting said cancer cell in vitro or in vivo with an amountof a compound of formula (I), effective to inhibit the growth of saidcancer cell.

The invention also provides a compound of formula (I) for use in medicaltherapy (preferably for use in treating cancer, e.g. solid tumors), aswell as the use of such compound for the manufacture of a medicamentuseful for the treatment of cancer and other diseases/disordersdescribed herein.

The invention further provides methods of treating inflammatory diseasesand disorders, including, for example, rheumatoid arthritis,osteoarthritis, allergies (such as asthma), and other inflammatoryconditions, such as pain (such as migraine), swelling, fever, psoriasis,inflammatory bowel disease, gastrointestinal ulcers, cardiovascularconditions, including ischemic heart disease and atherosclerosis,partial brain damage caused by stroke, skin conditions (eczema, sunburn,acne), leukotriene-mediated inflammatory diseases of lungs, kidneys,gastrointestinal tract, skin, prostatitis and paradontosis.

The invention further provides methods of treating immune responsedisorders, whereby the immune response is inappropriate, excessive orlacking. Such disorders include allergic responses, transplantrejection, blood transfusion reaction, and autoimmune disorders such assystemic lupus erythematosus and rheumatoid arthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effectiveness of parthenolide and derivatives of thepresent invention against prostate cancer cell line CWR22 in aclonogenic assay.

FIG. 2 shows the effectiveness of parthenolide and derivatives of thepresent invention against lung cancer cell line A-549 in a cellularproliferation MTS-PMS assay.

FIG. 3 shows the effectiveness of parthenolide and derivatives of thepresent invention against lung cancer cell line H-522 in a cellularproliferation MTS-PMS assay.

FIG. 4 shows the effectiveness of parthenolide and derivatives of thepresent invention against lung cancer cell line H-23 in a cellularproliferation MTS-PMS assay.

FIG. 5 shows the effectiveness of parthenolide and derivatives of thepresent. invention against lung cancer cell line H-460 in a cellularproliferation MTS-PMS assay.

FIG. 6 shows the effectiveness of parthenolide and derivatives of thepresent invention against breast cancer cell line HBL-100 in aclonogenic assay.

FIG. 7 shows the effectiveness of parthenolide and derivatives of thepresent invention against breast cancer cell line MD-231 in a clonogenicassay.

FIG. 8 shows the effectiveness of parthenolide and derivatives of thepresent invention against breast cancer cell line MD-436 in a clonogenicassay.

FIG. 9 shows parthenolide and DMAPT plasma concentrations at one hourfollowing oral gavage in mice.

FIG. 10 shows DMAPT dose-dependent inhibition of NF-kB DNA binding intwo transitional cell carcinoma cell lines HT-1376 and UMUC-3 inelectrophoretic mobility gel shift assay (EMSA).

FIG. 11 shows FSCScan analysis of TRAIL induced apoptosis assay usingMDA-MB-231 breast cancer cells treated first with DMAPT, thenTRAIL-RII-activating antibodies.

FIG. 12 shows FSCScan analysis of TRAIL induced apoptosis assay usingMDA-MB-231 breast cancer cells treated first with DMAPT, then TRAIL.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following definitions shall apply unless otherwiseindicated.

The phrase “optionally substituted” is used interchangeably with thephrase “substituted or unsubstituted.” Unless otherwise indicated, anoptionally substituted group may have a substituent at eachsubstitutable position of the group, and each substitution isindependent of any other. Also, combinations of substituents orvariables are permissible only if such combinations result in stablecompounds. In addition, unless otherwise indicated, functional groupradicals are independently selected. Where “optionally substituted”modifies a series of groups separated by commas (e.g., “optionallysubstituted A, B or C”; or “A, B or C optionally substituted with”), itis intended that each of the groups (e.g., A, B and C) is optionallysubstituted.

The term “aliphatic” or “aliphatic group” as used herein means astraight-chain or branched C₁₋₁₂ hydrocarbon chain that is completelysaturated or that contains one or more units of unsaturation, or amonocyclic C₃₋₈ hydrocarbon or bicyclic C₈₋₁₂ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic (also referred to herein as “carbocycle” or“cycloalkyl”), that has a single point of attachment to the rest of themolecule wherein any individual ring in said bicyclic ring system has3-7 members. For example, suitable aliphatic groups include, but are notlimited to, linear or branched or alkyl, alkenyl, alkynyl groups andhybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl.

The terms “alkyl,” “alkoxy,” “hydroxyalkyl,” “alkoxyalkyl” and“alkoxycarbonyl,” used alone or as part of a larger moiety include bothstraight and branched chains containing one to twelve carbon atoms. Theterms “alkenyl” and “alkynyl” used alone or as part of a larger moietyshall include both straight and branched chains containing two to twelvecarbon atoms.

The terms “haloalkyl,” “haloalkenyl” and “haloalkoxy” means alkyl,alkenyl or alkoxy, as the case may be, substituted with one or morehalogen atoms. The term “halogen” or “halo” means F, Cl, Br or I.

The term “heteroatom” means nitrogen, oxygen, or sulfur and includes anyoxidized form of nitrogen and sulfur, and the quaternized form of anybasic nitrogen. Heteroatom further includes Se, Si and P.

The term “aryl” used alone or in combination with other terms, refers tomonocyclic, bicyclic or tricyclic carbocyclic ring systems having atotal of five to fourteen ring members, wherein at least one ring in thesystem is aromatic and wherein each ring in the system contains 3 to 8ring members. The term “aryl” may be used interchangeably with the term“aryl ring”. The term “aralkyl” refers to an alkyl group substituted byan aryl. The term “aralkoxy” refers to an alkoxy group substituted by anaryl.

The term “heterocycloalkyl,” “heterocycle,” “heterocyclyl” or“heterocyclic” as used herein means monocyclic, bicyclic or tricyclicring systems having five to fourteen ring members in which one or morering members is a heteroatom, wherein each ring in the system contains 3to 7 ring members and is non-aromatic.

The term “heteroaryl,” used alone or in combination with other terms,refers to monocyclic, bicyclic and tricyclic ring systems having a totalof five to fourteen ring members, and wherein: 1) at least one ring inthe system is aromatic; 2) at least one ring in the system contains oneor more heteroatoms; and 3) each ring in the system contains 3 to 7 ringmembers. The term “heteroaryl” may be used interchangeably with the term“heteroaryl ring” or the term “heteroaromatic”. Examples of heteroarylrings include 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl,4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl,2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 1-pyrazolyl, 3-pyrazolyl,4-pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-pyrimidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,5-tetrazolyl, 2-triazolyl, 5-triazolyl, 2-thienyl, 3-thienyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, indazolyl, isoindolyl, acridinyl, andbenzoisoxazolyl. The term “heteroaralkyl” refers to an alkyl groupsubstituted by a heteroaryl. The term “heteroarylalkoxy” refers to analkoxy group substituted by a heteroaryl. \

An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) orheteroaryl (including heteroaralkyl, heteroarylalkoxy and the like)group may contain one or more substituents. Suitable substituents on anunsaturated carbon atom of an aryl, heteroaryl, aralkyl or heteroaralkylgroup are selected from halogen; haloalkyl; —CF₃; —R; —OR; —SR;1,2-methylenedioxy; 1,2-ethylenedioxy; protected OH (such as acyloxy);phenyl (Ph); Ph substituted with R; —O(Ph); —O—(Ph) substituted with R;—CH₂(Ph); —CH₂(Ph) substituted with R; —CH₂CH₂(Ph); —CH₂CH₂(Ph)substituted with R; —NO₂; —CN; —N(R)₂; —NRC(O)R; —NRC(O)N(R)₂; —NRCO₂R;—NRNRC(O)R; —NR—NRC(O)N(R)₂; —NRNRCO₂R; —C(O)C(O)R; —C(O)CH₂C(O)R;—CO₂R; —C(O)R; —C(O)N(R)₂; —OC(O)N(R)₂; —S(O)₂R; —SO₂N(R)₂; —S(O)R;—NRSO₂N(R)₂; —NRSO₂R; —C(═S)N(R)₂; —C(═NH)—N(R)₂; —(CH₂)_(y)NHC(O)R;—(CH₂)_(y)R; —(CH₂)_(y)NHC(O)NHR; —(CH₂)_(y)NHC(O)OR; —(CH₂)_(y)NHS(O)R;—(CH₂)_(y)NHSO₂R; or —(CH₂)_(y)NHC(O)CH((V)_(z)—R)(R) wherein each R isindependently selected from hydrogen, optionally substituted C₁₋₆aliphatic, an unsubstituted 5-6 membered heteroaryl or heterocyclicring, phenyl (Ph), —O(Ph), or —CH₂(Ph)—CH₂(Ph), wherein y is 0-6; z is0-1; and V is a linker group. When R is C₁₋₆ aliphatic, it may besubstituted with one or more substituents selected from —NH₂, —NH(C₁₋₄aliphatic), —N(C₁₋₄ aliphatic)₂, —S(O)(C₁₋₄ aliphatic), —SO₂(C₁₋₄aliphatic), halogen, (C₁₋₄ aliphatic), —OH, —O(C₁₋₄ aliphatic), —NO₂,—CN, —CO₂H, —CO₂(C₁₋₄ aliphatic), —O(halo C₁₋₄ aliphatic), or

-halo(C₁₋₄ aliphatic); wherein each C₁₋₄ aliphatic is optionally.

An aliphatic group or a non-aromatic heterocyclic ring may contain oneor more substituents. Suitable substituents on a saturated carbon of analiphatic group or of a non-aromatic heterocyclic ring are selected fromthose listed above for the unsaturated carbon of an aryl or heteroarylgroup and the following: ═O, ═S, ═NNHR, ═NN(R)₂, ═N—, ═NNHC(O)R,═NNHCO₂(alkyl), ═NNHSO₂(alkyl), or ═NR, where each R is independentlyselected from hydrogen or an optionally substituted C₁₋₆ aliphatic. WhenR is C₁₋₆ aliphatic, it may be substituted with one or more substituentsselected from —NH₂, —NH(C₁₋₄ aliphatic), —N(C₁₋₄ aliphatic)₂, halogen,—OH, —O—(C₁₋₄ aliphatic), —NO₂, —CN, —CO₂H, —CO₂(C₁₋₄ aliphatic),—O(halo C₁₋₄ aliphatic), or -halo(C₁₋₄ aliphatic); wherein each C₁₋₄aliphatic is optionally substituted.

Substituents on a nitrogen of a non-aromatic heterocyclic ring areselected from

—R, —N(R)₂, —C(O)R, —C(O)OR, —C(O)C(O)R, —C(O)CH₂C(O)R, —SO₂R,—SO₂N(R)₂, —C(═S)N(R)₂, —C(═NH)—N(R)₂ or —NRSO₂R; wherein each R isindependently selected from hydrogen, an optionally substituted C₁₋₆aliphatic, optionally substituted phenyl (Ph), optionally substituted—O(Ph), optionally substituted —CH₂(Ph), optionally substituted—CH₂CH₂(Ph), or an unsubstituted 5-6 membered heteroaryl or heterocyclicring. When R is a C₁₋₆ aliphatic group or a phenyl ring, it may besubstituted with one or more substituents selected from —NH₂, —NH(C₁₋₄aliphatic), —N(C₁₋₄ aliphatic)₂, halogen, —(C₁₋₄ aliphatic), —OH,—O—(C₁₋₄ aliphatic), —NO₂, —CN, —CO₂H, —CO₂(C₁₋₄ aliphatic), —O(haloC₁₋₄ aliphatic) or -halo(C₁₋₄ aliphatic); wherein each C₁₋₄ aliphatic isoptionally substituted.

The term “linker group” or “linker” means an organic moiety thatconnects two parts of a compound. Linkers include alkylidene chain thatis a saturated or unsaturated, straight or branched, C₁₋₈ carbon chainwhich is optionally substituted, and wherein up to two non-adjacentsaturated carbons of the chain are optionally replaced by R* wherein R*is —C(O)—, —C(O)C(O)—, —C(O)NR—, —C(O)NRNR—, —C(O)O—, —OC(O)—, —NRCO₂—,—O—, —NRC(O)NR—, —OC(O)NR—, —NRNR—, —NRC(O)—, —S—, —SO—, —SO₂—, —NR—,—SO₂NR—, or —NRSO₂—; wherein R is selected from hydrogen or optionallysubstituted aliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl,and preferably H or optionally substituted C₁₋₄ aliphatic. Optionalsubstituents on the alkylidene chain are as described above for analiphatic group. Alternatively, the linker group is R*.

The term “treatment” refers to any treatment of a pathologic conditionin a mammal, particularly a human, and includes: (i) preventing thepathologic condition from occurring in a subject which may bepredisposed to the condition but has not yet been diagnosed with thecondition and, accordingly, the treatment constitutes prophylactictreatment for the disease condition; (ii) inhibiting the pathologiccondition, i.e., arresting its development; (iii) relieving thepathologic condition, i.e., causing regression of the pathologiccondition; or (iv) relieving the conditions mediated by the pathologiccondition.

The term “therapeutically effective amount” refers to that amount of acompound of the invention that is sufficient to effect treatment, asdefined above, when administered to a mammal in need of such treatment.The therapeutically effective amount will vary depending upon thesubject and disease condition being treated, the weight and age of thesubject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art.

The term “pharmaceutically acceptable salts” includes, but is notlimited to, salts well known to those skilled in the art, for example,mono-salts (e.g. alkali metal and ammonium salts) and poly salts (e.g.di- or tri-salts) of the compounds of the invention. Pharmaceuticallyacceptable salts of compounds of formulas (I), (II), (III), or (IV) arewhere, for example, an exchangeable group, such as hydrogen in —OH,—NH—, or —P(═O)(OH)—, is replaced with a pharmaceutically acceptablecation (e.g. a sodium, potassium, or ammonium ion) and can beconveniently be prepared from a corresponding compound of formula (I)by, for example, reaction with a suitable base. In cases where compoundsare sufficiently basic or acidic to form stable nontoxic acid or basesalts, administration of the compounds as salts may be appropriate.Examples of pharmaceutically acceptable salts are organic acid additionsalts formed with acids that form a physiological acceptable anion, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, andα-glycerophosphate. Suitable inorganic salts may also be formed,including hydrochloride, sulfate, nitrate, bicarbonate, and carbonatesalts.

Pharmaceutically acceptable salts include quaternary ammonium saltsformed with WY; where Y is selected from halogen, tosylate,methanesulfonate, benzenesulfonate, trifluoromethanesulfonate and thelike; and R′ is selected from an optionally substituted cycloalkyl,heterocycloalkyl, aryl or heteroaryl.

Suitable acids include hydrofluoric acid, hydrochloric acid, hydrobromicacid, hydriodic acid, sulfuric acid, nitric acid, phosphoric acid,carbonic acid, boric acid, selenious acid, hydrogen sulfide,phosphomolybdic acid, phosphorous acid, sulfurous acid, citric acid,maleic acid, D-malic acid, L-lactic acid, D-lactic acid, DL-lactic acid,oxalic acid, methanesulfonic acid, valeric acid, oleic acid, lauricacid, para-toluenesulfonic acid, 1-naphthalensulfonic acid,2-naphthalensulfonic acid, phthalic acid, tartaric acid, L-malic acid,DL-malic acid, malonic acid, succinic acid, fumaric acid, glycolic acid,thioglycolic acid, glycine, sarcocine, sulfonic acid, nicotinic acid,picolinic acid, isonicotinic acid, benzoic acid and substituted benzoicacid where benzene ring bears one or more substituents.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example, by reacting asufficiently basic compound such as an amine with a suitable acidaffording a physiologically acceptable anion. Alkali metal (for example,sodium, potassium or lithium) or alkaline earth metal (for example,calcium) salts of carboxylic acids can also be made.

The term “prodrug” or “prodrugs” is used in its ordinary meaning in theart and means a compound of the invention that has its charged moietiesmasked or protected by another moiety that is designed to be cleavedunder particular physiological conditions, leaving the deprotected orunmasked compound of the invention. The use of masking agents is commonand well-known in the art and, in particular, masking phosphate orphosphonate groups. All such masking agents are suitable and can be usedwith the compounds of the invention. Various agents such as acyloxyalkyl esters are described by Srivasta et al., (1984 BioorganicChemistry 12, 118-12), and by Freeman et al. (1997 Progress in MedicinalChemistry 34:112-147) which are each incorporated in their entiretyherein by reference; and 3-phthalidyl phosphonate esters are describedby Dang Q., et al., (1999 Bioorganic & Med. Chem. Letters, 9:1505-1510),which is incorporated in its entirety herein by reference. For example,and not by way of limitation, Srivasta et al. also describeacetoxymethyl, isobutryloxymethyl, and pivaloxymethyl as masking agents.Other suitable masking groups comprising pivaloxyalkyl, e.g.,pivaloxymethyl, or a pivaloyloxy group as described by Farquhar D. etal., (1995 J. Med. Chem., 38:488-495) which is incorporated in itsentirety herein by reference. Still other masking or protecting agentsare described in U.S. Pat. Nos. 4,816,570 and 4,968,788 both of whichare incorporated in their entirety herein by reference. Lipid prodrugsare also suitable for use with the compounds of the invention. Bynon-limiting example, certain lipid prodrugs are described in Hostetleret al., (1997 Biochem. Pharm. 53:1815-1822), and Hostetler et al., 1996Antiviral Research 31:59-67), both of which are incorporated in theirentirety herein by reference. Additional examples of suitable prodrugtechnology is described in WO 90/00555; WO 96/39831; WO 03/095665A2;U.S. Pat. Nos. 5,411,947; 5,463,092; 6,312,662; 6,716,825; and U.S.Published Patent Application Nos. 2003/0229225 and 2003/0225277 each ofwhich is incorporated in their entirety herein by reference. Suchprodrugs may also possess the ability to target the drug compound to aparticular tissue within the patient, e.g., liver, as described by Erionet al., (2004 J. Am. Chem. Soc. 126:5154-5163; Erion et al., Am. Soc.Pharm. & Exper. Ther. DOI:10.1124/jept.104.75903 (2004); WO 01/18013 A1;U.S. Pat. No. 6,752,981), each of which is incorporated in theirentirety herein by reference. By way of non-limiting example, otherprodrugs suitable for use with the compounds of the invention aredescribed in WO 03/090690; and by Harris et al., (2002 Antiviral Chem &Chemo. 12: 293-300; Knaggs et al., 2000 Bioorganic & Med. Chem. Letters10: 2075-2078) each of which is incorporated in their entirety herein byreference.

The invention relates to the ability of the α-methylene-γ-butyrolactonemoiety in sesquiterpene lactones to be structurally modified by, forexample, Michael addition with amines or thiols. Modification of theparthenolide (1) molecule by this methodology using primary and/orsecondary amines to form water-soluble amino derivatives, affords amineadducts that can easily be obtained as different inorganic or organicsalts to further increase water solubility. Thus, a novel class of morewater-soluble parthenolide analogs is described. When compounds in thisclass were evaluated for antileukemic activity, it was found that thesecompounds were either equipotent as, or more potent than the parentcompound, parthenolide. More importantly, these novel analogs showedgreater cytotoxicity towards leukemia cells than towards normal cells.Thus, the present invention provides a new class of parthenolidederivatives with potent and selective anticancer activities.

In accordance with the present invention, there are provided compoundsof formula (I):

-   -   wherein:    -   X₁, X₂ and X₃ are heteroatoms;    -   R₄, R₅, R₆, R₇, R₈, R₉ and R₁₀ are independently selected from        H, halo, —OH, —NO₂, —CN and optionally substituted aliphatic,        cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and    -   Z is optionally substituted C₁₋₈ straight-chained or branched        aliphatic, optionally containing 1 or more double or triple        bonds, wherein one or more carbons are optionally replaced by R*        wherein R* is optionally substituted cycloalkyl,        heterocycloalkyl, aryl or heteroaryl; an amino acid residue, H,        —CN, —C(O)—, —C(O)C(O)—, —C(O)NR¹—, —C(O)NR¹NR²—, —C(O)O—,        —OC(O)—, —NR¹CO₂—, —O—, —NR¹C(O)NR²—, —OC(O)NR¹—, —NR¹NR²—,        —NR¹C(O)—, —S—, —SO—, —SO₂—, —NR¹—, —SO₂NR¹—, —NR¹R², or        —NR¹SO₂—, wherein R¹ and R² are independently selected from H        and optionally substituted aliphatic, cycloalkyl,        heterocycloalkyl, aryl or heteroaryl; or where R* is NR¹R², R¹        and R² optionally together with the nitrogen atom form an        optionally substituted 5-12 membered ring, said ring optionally        comprising 1 or more heteroatoms and/or a group selected from        —CO—, —SO—, —SO₂— and —PO—; or    -   a pharmaceutically acceptable salt, ester or prodrug thereof.

Presently preferred compounds include compounds of formula (I) whereinR₅, R₆, R₇, R₉ and R₁₀ are independently selected from H, halo, —OH,—NO₂, —CN, —CH₃, —CF₃, —CH₂CH₃, —CH₂CF₃, —CH₂Cl, —CH₂OH, —CH₂CH₂OH and—CH₂NH₂. Further preferred embodiments include compounds where R₅, R₆,R₇, R₉ and R₁₀ are each H.

Other preferred embodiments of the present invention include compoundswhere R₄ and R₈ are independently selected from optionally substitutedC₁-C₄ alkyl. In one preferred embodiment, R₄ is —CH₃, and in another, R₄and R₈ are each —CH₃.

In one embodiment X₁, X₂ and X₃ are heteroatoms independently selectedfrom O, N and S, and in one particular embodiment, X₁, X₂ and X₃ areeach O.

According to one embodiment, it is preferred that where R₅, R₆, R₇, R₉and R₁₀ are H, R₄ and R₈ are each —CH₃, and X₁, X₂ and X₃ are each O; Zis not ═CH₂.

According to a further embodiment of the invention, Z is—(CH₂)_(m)—NR¹R² where m is an integer from 0 to 4, where preferably,R₅, R₆, R₇, R₉, and R₁₀ are H; and R₄ and R₈ are each —CH₃. In oneparticular embodiment, m is 1. In other embodiments, R¹ and R² areindependently selected from hydrogen, —CN or optionally substitutedC₁-C₄ alkyl. In particular embodiments, R¹ and R² are independentlyselected from —NO₂, —CN, —CH₃, —CF₃, —CH₂CH₃, —CH₂CF₃, —CH₂Cl, —CH₂OH,—CH₂CH₂OH and —CH₂NH₂.

In yet a further embodiment, R¹ and R² together with N form anoptionally substituted ring. The ring is a monocyclic, bicyclic ortricyclic aliphatic or aryl ring system, where the ring system isoptionally substituted and optionally comprises one or more heteroatomsor a group selected from —CO—, —SO—, —SO₂— and —PO—. In one particularembodiment, R¹ and R² are —CH₂(CH₂)_(n)CH₂Y—, where Y is a heteroatom ora group selected from —CO—, —SO—, —SO₂— and —PO—; n is an integer 0 to5; and together with N form an optionally substituted ring, which may beadditionally fused to a cycloalkyl or aryl group to form a bicyclic ortricyclic ring system, where the system is optionally substituted andoptionally comprises one or more heteroatoms. Alternatively, R₁ and R₂are —(CH₂)_(a)—Y—(CH₂)_(b)—, where Y is a heteroatom or a group selectedfrom —CO—, —SO—, —SO₂— and —PO—; a is an integer 0 to 5; b is an integer0 to 5; where the sum of a and b is 0 to 5; and together with N form anoptionally substituted ring, the ring being optionally fused to acycloalkyl or aryl group to form a bicyclic or tricyclic ring system,where the system is optionally substituted and optionally comprises oneor more heteroatoms.

Examples of ring systems include an optionally substituted uracil ringor a derivative thereof. Other examples include optionally substitutepyrrole, imidazole, purine and pyrazole and derivative thereof. Examplesof fused ring systems include optionally substituted aziridin-1-yl,azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, homopiperidyn-1-yl andheptamethyleneimin-1-yl.

According to a further embodiment of the invention, Z is—(CH₂)_(m)—S(O)_(n)—R¹ where m is an integer from 0 to 4, n is aninteger from 0 to 2, where preferably, R₅, R₆, R₇, R₉, and R₁₀ are H;and R₄ and R₈ are each —CH₃. In one particular embodiment, m is 1. Inanother embodiment n is 0. In other embodiments, R¹ is independentlyselected from hydrogen, —CN, optionally substituted C₁-C₄ alkyl or aryl.In particular embodiments, R¹ is selected from —C₆H₅, —CH₂CH(CO₂H)(NH₂),and —CH₂CO₂H.

Exemplary compounds of the present invention include:

-   11S,11,13-Dihydro,13-dimethylaminoparthenolide (DMAPT);-   11S,11,13-Dihydro,13-diethylaminoparthenolide;-   11S,11,13-Dihydro,13-(tert-butylamino)parthenolide;-   11S,11,13-Dihydro,13-(pyrrolidin-1-yl)parthenolide;-   11S,11,13-Dihydro,13-(piperidin-1-yl)parthenolide (PIPT);-   11S,11,13-Dihydro,13-(morpholin-1-yl)parthenolide;-   11S,11,13-Dihydro,13-(4-methylpiperidin-1-yl)parthenolide (4MEPT);-   11S,11,13-Dihydro,13-(4-methylpiperazin-1-yl)parthenolide;-   11S,11,13-Dihydro,13-(homopiperidin-1-yl)parthenolide;-   11S,11,13-Dihydro,13-(heptamethyleneimin-1-yl)parthenolide;-   11S,11,13-Dihydro,13-(azetidin-1-yl)parthenolide;-   11S,11,13-Dihydro,13-methylbutyl aminoparthenolide;-   11S,11,13-Dihydro,13-methyl pentyl aminoparthenolide;-   11S,11,13-Dihydro,13-ethylaminoparthenolide;-   11S,11,13-Dihydro,13-methylaminoparthenolide;-   11S,11,13-Dihydro,13-cyclopropylaminoparthenolide;-   11S,11,13-Dihydro,13-propargylaminoparthenolide;-   11S,11,13-Dihydro,13-(N-benzyl-N-ethylamine)parthenolide;-   11S,11,13-Dihydro,13-(N-prolyl)parthenolide;-   11S,11,13-Dihydro,13-(S-thiophenolyl)parthenolide;-   11S,11,13-Dihydro,13-(N,N-diethanolamine)parthenolide-   11S,11,13-Dihydro,13-(thiomorpholin-4-yl)parthenolide;-   11S,11,13-Dihydro,13-(4-hydroxypiperidin-1-yl)parthenolide;-   11S,11,13-Dihydro,13-(1-methylhomopiperizin-4-yl)parthenolide;-   11S,11,13-Dihydro,13-(S-mercaptoacetyl)parthenolide;-   11S,11,13-Dihydro,13-(4-(2′-hydroxyethyl)piperidin-1-yl)parthenolide;-   11S,11,13-Dihydro,13-(piperazin-1-yl-4-carboxaldehyde)parthenolide;-   11S,11,13-Dihydro,13-(4-benzylpiperidin-1-yl)parthenolide;-   11S,11,13-Dihydro,13-(piperidin-1-yl-4-carboxylic acid)parthenolide;-   11S,11,13-Dihydro,13-(azetidin-1-yl-3-carboxylic acid)parthenolide;-   11S,11,13-Dihydro,13-(S-cysteinyl)parthenolide;-   11S,11,13-Dihydro,13-(4-(piperidin-1′-yl)piperidin-1-yl))parthenolide;    and-   11S,11,13-Dihydro,13-diallylaminoparthenolide.

Those of skill in the art will recognize that the invention comprisescompounds that may contain one or more chiral centers on, for example,the parthenolide C-11 and thus can exist as racemic mixtures as purediastereomers, or as pure enantiomers. For many applications, it ispreferred to carry out stereoselective synthesis and/or to subject thereaction product to appropriate purification steps so as to producesubstantially stereochemically pure or optically pure materials.Suitable stereoselective synthetic procedures for producingstereochemically pure or optically pure materials are well known in theart, as are procedures for resolving racemic mixtures into theiroptically pure enantiomers.

The present invention further provides for compounds having formula (I),or a pharmaceutically acceptable salt thereof, wherein Z is a group thatis converted to ═CH₂ under physiological conditions during or afteradministration to a mammalian patient, thereby yielding a methylenegroup. In particular embodiments X₁, X₂ and X₃ are O; R₅, R₆, R₇, R₉,and R₁₀ are H; R₄ and R₈ are —CH₃; and Z is optionally substituted C₁₋₈straight-chained or branched aliphatic, optionally containing 1 or moredouble or triple bonds, wherein one or more carbons are optionallyreplaced by R* wherein R* is optionally substituted cycloalkyl,heterocycloalkyl, aryl or heteroaryl; an amino acid residue, H, —CN,—C(O)—, —C(O)C(O)—, —C(O)NR¹—, —C(O)NR¹NR²—, —C(O)O—, —OC(O)—, —NR¹CO₂—,—O—, —NR¹C(O)NR²—, —OC(O)NR¹—, —NR¹NR²—, —NR¹C(O)—, —S—, —SO—, —SO₂—,—NR¹—, —SO₂NR¹—, —NR¹R², or —NR¹SO₂—, wherein R¹ and R² areindependently selected from H and optionally substituted aliphatic,cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In preferredembodiments Z is —CH₂N(CH₃)₂, —CH₂N(H)(C(CH₃)₃), —CH₂N(CH₂CH₃)₂,—CH₂N(CH₂CH═CH₂)₂, —CH₂-azetidine, —CH₂-pyrrolidine, —CH₂-piperidine,—CH₂-homopiperidine, —CH₂-heptamethyleneimine, —CH₂-4-methylpiperidine,—CH₂-morpholine, —CH₂-pyrrolidine, —CH₂-proline, —CH₂-thiophenol,—CH₂-diethanolamine, —CH₂-hydroxypiperidine, —CH₂-methylhomopiperazine,—CH₂-thiomorpholine, —CH₂-mercaptoacetic acid, —CH₂-benzylpiperidine,—CH₂-piperidine-4-carboxylic acid, —CH₂-azetidine-3-carboxylic acid,—CH₂-piperidinylpiperidine, or —CH₂-cysteine.

The present invention further provides for compounds having formula (I),or a pharmaceutically acceptable salt thereof, wherein Z is CH₂N(CH₃)₂which under physiological conditions during or after administration to amammalian patient, undergoes mono- or di-demethylation; conversion to═CH₂, or cysteine or protein conjugation. In particular embodiments, X₁,X₂ and X₃ are O; R₅, R₆, R₇, R₉, and R₁₀ are H; R₄ and R₈ are —CH₃;

The present invention further provides compounds wherein theparthenolide based derivatives of formula (I) form dimers or duplexeswith another molecule of formula (I) or with basic nitrogen-containingsynergistic anticancer drug molecules such as 5-fluorouracil,cytarabine, mytomycin C, Doxorubicin and Daunorubicin. Accordingly, thepresent invention provides compounds of the general formula (II):

-   -   where L is a linker, as defined above, and W is a molecule with        anti-cancer growth activity. Where W is another parthenolide        derivative of formula (I), the present invention provides        compounds of formula (III):

-   -   where R′₄, R′₅, R′₆, R′₇, R′₈, R′₉, R′₁₀, X′₁, X′₂ and X′₃ are        independently as defined above for their counterparts, R₆, R₇,        R₈, R₉, R₁₀, X₁, X₂ and X₃; and L is a linker, as defined above.        Preferred linkers include optionally substituted alkyl and amine        groups. In one particular embodiment, L is —CH₂N(R)CH₂—, where R        is as defined above. Included are pharmaceutically acceptable        salts formed with inorganic and/or organic acids, as defined        above for compounds of formula (I).

In accordance with another embodiment of the invention, the methods forthe preparation of the amino analogs described in this invention aredisclosed in Schemes I and II.

In the above scheme, the solvent is selected from a low alkyl alcohol,such as methanol, ethanol, propanol, isopropanol, n-butanol,tert-butanol, and chloroform, methylene chloride, benzene, toluene,tetrahydrofuran, dioxane, 1,2-dimethoxyethane, pyridine, carbontetrachloride, diethyl ether, tert-butyl methyl ether and/or the mixtureof two or more of the solvents listed above. The base is selected from alow trialkylamine, such as trimethylamine, triethylamine,tripropylamine, and tributylamine, and pyridine, 2-, 3-, and4-picolines, 2-, 3-, and 4-dimethylaminopyridines. The temperature isselected from −20° C. to 130° C. The reaction time required to effectthe desired coupling reaction can vary widely, typically falling in therange of 30 min to 24 hours. Purification can be achieved by a varietyof techniques, such as, liquid chromatography through neutral or basicsilica gel, bonded silica gel phases such as octadecylsilica,octylsilica and the like, cellulose or alumina with the solvent such as,for example, the mixture of chloroform and methanol or ethanol, themixture of methylene chloride and methanol or ethanol, the mixture ofhexane and acetone or acetonitrile or methanol or ethanol orisopropanol, the mixture of diethyl ether and acetone or acetonitrile ormethanol or ethanol or isopropanol; and recrystallization using normalorganic solvent or solvent mixture, such as methanol, ethanol, propanol,isopropanol, tert-butanol, acetonitrile, diethyl ether, chloroform,methylene chloride and the mixture of two or more solvents listed above.The purity of the invention compounds prepared is assessed by massspectrometry, nuclear magnetic resonance spectrometry (NMR) andelemental combustion analysis.

Furthermore, in accordance with still another embodiment of the presentinvention, the methods for the preparation of the invention salts aredisclosed in Schemes III and IV.

In these schemes, HX is selected from hydrochloride, hydrobromide,hydroiodide, perchlorate, sulfate, hemisulfate, mesylate,toluenesulfonate, benzenesulfonate, succinate, hemisuccinate, fumarate,tartarate, ascorbate, acetate, hemifumarate, maleate, citrate, oxalate,malonate, malic, propionate and benzoate; Yθ is selected from halide(fluoride, chloride, bromide, iodide), methylsulfonate,toluenesulfonate, benzenesulfonate and sulfate; and the solvent isselected from a low alkyl alcohol, such as diethyl ether, methanol,ethanol, propanol, isopropanol, n-butanol, tert-butanol, and chloroform,methylene chloride, benzene, toluene, tetrahydrofuran, dioxane,1,2-dimethoxyethane, pyridine, carbon tetrachloride, tert-butyl methylether, acetone and/or the mixture of two or more of the solvents listedabove. The temperature is selected from −20° C. to 50° C. Purificationcan be achieved by recrystallization using normal organic solvent orsolvent mixture, such as methanol, ethanol, acetone, propanol,isopropanol, t-butanol, acetonitrile, diethyl ether, chloroform,methylene chloride and the mixture of two or more solvents listed above.

The present invention further provides analogues of compounds of formula(I). Examples are described below and include costunolide,dehydrocostuslactone, alantolactone, isoalantolactone,amino-3-oxo-isoalantolactone, helenalin, 11,13-dihydrohelenalin,aminocyanaropicrin, aminodesacylcyanaropicrin, (+)-aminoreyonosin,aminosantamarin, aminosoulangianolide and aminoisotelekin. In addition,the present invention provides compounds of the analogues below,amino-3-oxo-isoalantolactone, aminocyanaropicrin,aminodesacylcyanaropicrin, (+)-aminoreyonosin, aminosantamarin,aminosoulangianolide and aminoisotelekin wherein (—CH₂—NR₁R₂) isreplaced by Z, where Z is as defined herein.

The invention relates to the ability of the α-methylene-γ-butyrolactonemoiety in all the above-mentioned sesquiterpene lactones to bestructurally modified by, for example, Michael addition with thefollowing chemical entities to form more water-soluble derivatives thanthe corresponding parent sesquiterpene, and with improved propertiesconducive for drug development, such as, chemical stability, reducedtoxicity and oral bioavailability.

The present invention further provides compounds of theparthenolide-analog dehydrocostuslactone based derivatives of formula(IV):

Presently preferred compounds include compounds of formula (IV) whereinR₃, R₅, R₆, R₉ and R₁₀ are independently selected from H, halo, —OH,—NO₂, —CN, —CH₃, —CF₃, —CH₂CH₃, —CH₂CF₃, —CH₂Cl, —CH₂OH, —CH₂CH₂OH and—CH₂NH₂. Further preferred embodiments include compounds where R₃, R₅,R₆, R₉ and R₁₀ are each H.

Other preferred embodiments of the present invention include compoundswhere R₄ and R₈ are independently selected from optionally substitutedC₁-C₄ alkyl. In one preferred embodiment, R₄ and R₈ are each ═CH₂.

In one embodiment X₁ and X₂ are heteroatoms independently selected fromO, N and S, and in one particular embodiment, X₁ and X₂ are each O.

According to a further embodiment of the invention, Z is—(CH₂)_(m)—NR¹R² where m is an integer from 0 to 4, where preferably,R₅, R₆, R₇, R₉ and R₁₀ are H; and R₄ and R₈ are each —CH₃. In oneparticular embodiment, m is 1. In other embodiments, R¹ and R² areindependently selected from hydrogen, —CN or optionally substitutedC₁-C₄ alkyl. In particular embodiments, R¹ and R² are independentlyselected from —NO₂, —CN, —CH₃, —CF₃, —CH₂CH₃, —CH₂CF₃, —CH₂Cl, —CH₂OH,—CH₂CH₂OH and —CH₂NH₂.

In yet a further embodiment, R¹ and R² together with N form anoptionally substituted ring. The ring is a monocyclic, bicyclic ortricyclic aliphatic or aryl ring system, where the ring system isoptionally substituted and optionally comprises one or more heteroatomsor a group selected from —CO—, —SO—, —SO₂— and —PO—. In one particularembodiment, R¹ and R² are —CH₂(CH₂)_(n)CH₂Y—, where Y is a heteroatom ora group selected from —CO—, —SO—, —SO₂— and —PO—; n is an integer 0 to5; and together with N form an optionally substituted ring, which may beadditionally fused to a cycloalkyl or aryl group to form a bicyclic ortricyclic ring system, where the system is optionally substituted andoptionally comprises one or more heteroatoms. Alternatively, R₁ and R₂are —(CH₂)_(n)—Y—(CH₂)_(b)—, where Y is a heteroatom or a group selectedfrom —CO—, —SO—, —SO₂— and —PO—; a is an integer 0 to 5; b is an integer0 to 5; where the sum of a and b is 0 to 5; and together with N form anoptionally substituted ring, the ring being optionally fused to acycloalkyl or aryl group to form a bicyclic or tricyclic ring system,where the system is optionally substituted and optionally comprises oneor more heteroatoms.

Examples of ring systems include an optionally substituted uracil ringor a derivative thereof. Other examples include optionally substitutepyrrole, imidazole, purine and pyrazole and derivative thereof. Examplesof fused ring systems include optionally substituted aziridin-1-yl,azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, homopiperidyn-1-yl andheptamethyleneimin-1-yl.

With respect to formulas (I) and (IV), in other embodiments, Z ishydroxylamine, a hydroxyalkylamino compound, a thioalkylamino compound,a diaminoalkane. Examples include ethylenediamine, piperazine,triaminoalkanes, polyamines, polylysine, putrescine, spermine,spermidine, aminoguanidines and agmatine. In other embodiments, Z is anamino acid. For example glycine, serine, hydroxyproline, β-alanine,cysteine, homocysteine, arginine, lysine, glutamic acid, ornithine,aspartic acid, γ-aminobutyric acid, or taurine. In other embodiments, Zis an amino sugar; for example glucosamine. In other embodiments, Z is apolyoxyethylene glycol of various molecular weights, each of whichterminate in an amino functionality that will form an adduct with theappropriate sesquiterpene.

Modification of the sesquiterpene molecules by these methodologies,affords adducts that can easily be obtained as different inorganic ororganic salts to further increase water solubility.

The compounds described herein are useful for treating cancer. Cancerstreatable by the present therapy include the solid and hematologicaltumors, such as prostate cancer, ovarian cancer, breast cancer, braincancer and hepatic cancer, comprising administering to a mammalafflicted with said cancer an amount of parthenolide derivativeeffective to inhibit the viability of cancer cells of said mammal. Theparthenolide derivative may be administered as primary therapy, or asadjunct therapy, either following local intervention (surgery,radiation, local chemotherapy) or in conjunction with at least one otherchemotherapeutic agent discussed hereinabove, as well as the solidtumors disclosed in U.S. Pat. No. 5,514,555. Hematological cancers, suchas the leukemias are disclosed in the Mayo Clinic Family Health Book, D.E. Larson, ed., William Morrow, N.Y. (1990) and include CLL, ALL, CMLand the like. Compounds of the present invention may be used in bonemarrow transplant procedure to treat bone marrow prior to reintroductionto the patient. In addition, the compounds of the present invention maybe used as chemotherapy sensitizers or radiation therapy sensitizers.Accordingly, a patient, or cells, or tissues, derived from a cancerpatient, are pre-treated with the compounds prior to standardchemotherapy or radiation therapy. The present invention contemplatesthat parthenolide may also be used in such methods.

Within another aspect of the present invention, methods are provided forinhibiting angiogenesis in patients with non-tumorigenic,angiogenesis-dependent diseases, comprising administering atherapeutically effective amount of a composition comprisingparthenolide derivative to a patient with a non-tumorigenicangiogenesis-dependent disease, such that the formation of new bloodvessels is inhibited. Within other aspects, methods are provided forinhibit reactive proliferation of endothelial cells or capillaryformation in non-tumorigenic, angiogenesis-dependent diseases, such thatthe blood vessel is effectively occluded. Within one embodiment, theanti-angiogenic composition comprising parthenolide derivative isdelivered to a blood vessel which is actively proliferating andnourishing a tumor.

In addition to tumors, numerous other non-tumorigenicangiogenesis-dependent diseases, which are characterized by the abnormalgrowth of blood vessels, may also be treated with the anti-angiogenicparthenolide derivative compositions, or anti-angiogenic factors of thepresent invention. Anti-angiogenic parthenolide derivative compositionsof the present invention can block the stimulatory effects ofangiogenesis promoters, reducing endothelial cell division, decreasingendothelial cell migration, and impairing the activity of theproteolytic enzymes secreted by the endothelium. Representative examplesof such non-tumorigenic angiogenesis-dependent diseases include cornealneovascularization, hypertrophic scars and keloids, proliferativediabetic retinopathy, arteriovenous malformations, atheroscleroticplaques, delayed wound healing, hemophilic joints, nonunion fractures,Osler-Weber syndrome, psoriasis, pyogenic granuloma, scleroderma,trachoma, menorrhagia, retrolental fibroplasia and vascular adhesions.The pathology and treatment of these conditions is disclosed in detailin published PCT application PCT/CA94/00373 (WO 95/03036), at pages26-36. Topical or directed local administration of the presentcompositions is often the preferred mode of administration oftherapeutically effective amounts of parthenolide derivative, i.e., indepot or other controlled release forms.

Anti-angiogenic compositions of the present invention may also beutilized in a variety of other manners. For example, they may beincorporated into surgical sutures in order to prevent stitchgranulomas, implanted in the uterus (in the same manner as an IUD) forthe treatment of menorrhagia or as a form of female birth control,administered as either a peritoneal lavage fluid or for peritonealimplantation in the treatment of endometriosis, attached to a monoclonalantibody directed against activated endothelial cells as a form ofsystemic chemotherapy, or utilized in diagnostic imaging when attachedto a radioactively labelled monoclonal antibody which recognizes activeendothelial cells. The magnitude of a prophylactic or therapeutic doseof parthenolide derivative, an analog thereof or a combination thereof,in the acute or chronic management of cancer, i.e., prostate or breastcancer, will vary with the stage of the cancer, such as the solid tumorto be treated, the chemotherapeutic agent(s) or other anti-cancertherapy used, and the route of administration. The dose, and perhaps thedose frequency, will also vary according to the age, body weight, andresponse of the individual patient. In general, the total daily doserange for parthenolide derivative and its analogs, for the conditionsdescribed herein, is from about 0.5 mg to about 2500 mg, in single ordivided doses. Preferably, a daily dose range should be about 1 mg toabout 100 mg, in single or divided doses, most preferably about 5-50 mgper day. In managing the patient, the therapy should be initiated at alower dose and increased depending on the patient's global response. Itis further recommended that infants, children, patients over 65 years,and those with impaired renal or hepatic function initially receivelower doses, and that they be titrated based on global response andblood level. It may be necessary to use dosages outside these ranges insome cases. Further, it is noted that the clinician or treatingphysician will know how and when to interrupt, adjust or terminatetherapy in conjunction with individual patient response. The terms “aneffective amount” or “an effective sensitizing amount” are encompassedby the above-described dosage amounts and dose frequency schedule.

Any suitable route of administration may be employed for providing thepatient with an effective dosage of parthenolide derivative (e.g., oral,sublingual, rectal, intravenous, epidural, intrethecal, subcutaneous,transcutaneous, intramuscular, intraperitoneal, intracutaneous,inhalation, transdermal, nasal spray, nasal gel or drop, and the like).While it is possible that, for use in therapy, parthenolide derivativeor its analogs may be administered as the pure chemicals, as byinhalation of a fine powder via an insufflator, it is preferable topresent the active ingredient as a pharmaceutical formulation. Theinvention thus further provides a pharmaceutical formulation comprisingparthenolide derivative or an analog thereof, together with one or morepharmaceutically acceptable carriers therefor and, optionally, othertherapeutic and/or prophylactic ingredients. The carrier(s) must be‘acceptable’ in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof, such asa human patient or domestic animal.

Pharmaceutical formulations include those suitable for oral orparenteral (including intramuscular, subcutaneous and intravenous)administration. Forms suitable for parenteral administration alsoinclude forms suitable for administration by inhalation or insufflationor for nasal, or topical (including buccal, rectal, vaginal andsublingual) administration. The formulations may, where appropriate, beconveniently presented in discrete unit dosage forms and may be preparedby any of the methods well known in the art of pharmacy. Such methodsinclude the step of bringing into association the active compound withliquid carriers, solid matrices, semi-solid carriers, finely dividedsolid carriers or combinations thereof, and then, if necessary, shapingthe product into the desired delivery system.

Pharmaceutical formulations suitable for oral administration may bepresented as discrete unit dosage forms such as hard or soft gelatincapsules, cachets or tablets each containing a predetermined amount ofthe active ingredient; as a powder or as granules; as a solution, asuspension or as an emulsion; or in a chewable base such as a syntheticresin or chicle for ingestion of the agent from a chewing gum. Theactive ingredient may also be presented as a bolus, electuary or paste.Tablets and capsules for oral administration may contain conventionalexcipients such as binding agents, fillers, lubricants, disintegrants,or wetting agents. The tablets may be coated according to methods wellknown in the art, i.e., with enteric coatings.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may contain conventionaladditives such as suspending agents, emulsifying agents, non-aqueousvehicles (which may include edible oils), or preservatives.

The compounds according to the invention may also be formulated forparenteral administration (e.g., by injection, for example, bolusinjection or continuous infusion) and may be presented in unit dose formin ampules, pre-filled syringes, small volume infusion containers or inmulti-dose containers with an added preservative. The compositions maytake such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

For topical administration to the epidermis, the compounds may beformulated as ointments, creams or lotions, or as the active ingredientof a transdermal patch. Suitable transdermal delivery systems aredisclosed, for example, in A. Fisher et al. (U.S. Pat. No. 4,788,603),or R. Bawa et al. (U.S. Pat. Nos. 4,931,279; 4,668,506 and 4,713,224).Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents.

Formulations suitable for topical administration in the mouth includeunit dosage forms such as lozenges comprising active ingredient in aflavored base, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert base such as gelatin andglycerin or sucrose and acacia; mucoadherent gels, and mouthwashescomprising the active ingredient in a suitable liquid carrier.

When desired, the above-described formulations can be adapted to givesustained release of the active ingredient employed, e.g., bycombination with certain hydrophilic polymer matrices, e.g., comprisingnatural gels, synthetic polymer gels or mixtures thereof. The polymermatrix can be coated onto, or used to form, a medical prosthesis, suchas a stent, valve, shunt, graft, or the like.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the active compound with thesoftened or melted carrier(s) followed by chilling and shaping in molds.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

For administration by inhalation, the compounds according to theinvention are conveniently delivered from an insufflator, nebulizer or apressurized pack or other convenient means of delivering an aerosolspray. Pressurized packs may comprise a suitable propellant such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecompounds according to the invention may take the form of a dry powdercomposition, for example, a powder mix of the compound and a suitablepowder base such as lactose or starch. The powder composition may bepresented in unit dosage form in, for example, capsules or cartridgesor, e.g., gelatin or blister packs from which the powder may beadministered with the aid of an inhalator or insufflator.

For intra-nasal administration, the compounds of the invention may beadministered via a liquid spray, such as via a plastic bottle atomizer.Typical of these are the Mistometer® (Wintrop) and the Medihaler®(Riker).

For topical administration to the eye, the compounds can be administeredas drops, gels (U.S. Pat. No. 4,255,415), gums (see U.S. Pat. No.4,136,177) or via a prolonged-release ocular insert.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES Example 1 General Synthetic Procedure for the Preparation of11S,11,13-Dihydro,13-Substituted Aminoparthenolides

A mixture of parthenolide (Sigma P 0667, 100 mg, 0.4 mmol), theappropriate primary amine or secondary amine (2 mmol), and triethylamine(1 to 2 mL) in 30 mL of anhydrous ethanol was stirred at a specifictemperature ranging from ambient temperature to the temperature of therefluxing solvent utilized, or was left to stand in the refrigerator(−20° C. to 4° C.) overnight for 24 hours. Ethanol, triethylamine and/orthe appropriate volatile amine were then evaporated under vacuum in arotary evaporator. The resulting residue was subjected to silica gelcolumn chromatographic purification using chloroform-methanol ormethylene chloride-methanol mixed solvent as the mobile phase. NMR(Varian, 300 MHz and 400 MHz) and GC/MS (Agilent, 6890GC and 5973MSD)analysis methodologies were utilized to assure the identity and purityof the synthetic compounds.

Example 2 11S,11,13-Dihydro,13-dimethylaminoparthenolide (DMAPT)

Parthenolide (100 mg, 0.4 mmol), dimethylamine (2M in methanol, 1 mL),triethylamine (2 mL), ethanol (30 mL) were refluxed overnight. Aftercolumn purification, 109 mg of pale yellow11S,11,13-dihydro,13-dimethylaminoparthenolide was obtained (Yield:93%). Melting point: 143-144° C. ¹H-NMR (300 MHz, CDCl₃): δ 5.22 (1H,d), 3.85 (1H, t), 2.75 (2H, m), 2.65 (1H, dd), 2.5-2.3 (3H, m), 2.25(6H, s), 2.5-2.0 (5H, m), 1.7 (3H, s), 1.3 (3H, s), 1.3-1.15 (1H, m).¹³C-NMR (300 MHz, CDCl₃): δ 176.1, 134.4, 124.8, 81.9, 66.4, 61.3, 57.6,47.8, 46.4, 46.1, 41.0, 36.6, 29.9, 24.0, 17.2, 16.9. Mass Spec (GC-MS):293 (M⁺) Retention time: 12.56 minutes. Ultra-violet (Methanol): λ_(max)at 214 nm. Infra-Red (Nujol): 1757.9, 1460, 1377 cm⁻¹. X-raycrystallographic analysis using a Nonius KappaCCD diffractometer DMAPT(11S,11,13-dihydro, 13-dimethylaminoparthenolide) has theS-configuration at C-11.

Example 3 11S,11,13-Dihydro,13-diethylaminoparthenolide

Parthenolide (100 mg, 0.4 mmol), diethylamine (200 mg, 2.7 mmol),triethylamine (2 mL), ethanol (30 mL) were refluxed overnight. Aftercolumn purification, 114 mg of yellow11S,11,13-dihydro,13-diethylaminoparthenolide was obtained (Yield: 88%).

Example 4 Preparation of Salts of 11S,11,13-Dihydro,13-aminoparthenolideDerivatives

The aminoparthenolide derivative was dissolved in anhydrous ether and tothis solution was added the corresponding acid in ether or ethanol. Themixture was kept in the refrigerator (4° C.) overnight. The crystalsformed was filtered and dried under vacuum, or submitted to furtherrecrystallization, if needed.

Example 5 Preparation of11S,11,13-dihydro,13-(piperidin-1-yl)parthenolide hydrochloride

11S,11,13-dihydro,13-(piperidin-1-yl)parthenolide (5 mg) was dissolvedin 2 mL of dry ether. Hydrochloride in ether (1M, 0.015 mL) was added tothe ether solution until the solution became cloudy; then more ether wasadded and the mixture was heated to obtain a clear solution. The mixturewas left in refrigerator (4° C.) for more than 24 hours. The whitecrystals that formed were filtered through filter paper, and dried undervacuum overnight (Yield: 18%).

Example 6 Preparation of 11S,11,13-dihydro,13-dimethylaminoparthenolidemaleate

To 11S,11,13-dihydro,13-dimethylaminoparthenolide (30 mg, 0.1 mmol) inanhydrous ethanol (5 mL) was added maleic acid (12 mg, 0.1 mmol) in 3 mLof anhydrous ethanol. The solution was shaken well and filtered througha regular filter paper. The clear solution was left in the refrigeratorfor a week. The white crystals formed were obtained by filtration, driedin a desiccator under vacuum with anhydrous CaCl₂ (Yield: 55%).

Example 7 Preparation of 11S,11,13-dihydro,13-Dimethylaminoparthenolidemethiodide

To 11S,11,13-dihydro,13-dimethylaminoparthenolide (30 mg, 0.1 mmol) inanhydrous methanol (5 mL) was added iodomethane (90 mg, 0.6 mmol) inmethanol (1 mL). The clear solution was shaken and stored at roomtemperature. After three days, the methanol was evaporated, the paleyellow residue was dried in a desiccator under vacuum, over anhydrousCaCl₂. Recrystallization from acetone-ether afforded pale yellowcrystals (Yield: 86%).

Example 8 11S,11,13-dihydro,13-(4-Methylpiperidin-1-yl)parthenolidemethiodide

To 11S,11,13-dihydro,13-(4-methylpiperidin-1-yl)parthenolide (35 mg, 0.1mmol) in anhydrous methanol (5 mL) was added iodomethane (90 mg, 0.6mmol) in methanol (1 mL1). The clear solution was shaken and stored atroom temperature. After three days, the methanol was evaporated, thepale yellow residue was dried in a desiccator under vacuum, overanhydrous CaCl₂. Recrystallization from acetone-ether afforded paleyellow crystals (Yield: 79%).

Example 9 Assay for Antileukemic Activity

For apoptosis analysis, one million primary acute myelogenous leukemia(AML) cells were washed with cold PBS and resuspended in 200 microlitersof Annexin binding buffer (10 mM HEPES/NaOH pH 7.4; 140 mM NaCl; 2.5 mMCaCl₂). Annexin V-FITC (Pharmingen) and 0.25 mg/mL 7-AAD(7-aminoactinomycin D, Molecular Probes, CA) were added and the tubeswere incubated at room temperature in the dark for 15 minutes. Cellswere then diluted with 200 microliters of Annexin binding buffer andanalyzed immediately by flow cytometry. Viable cells were identified asfailing to label with Annexin V or 7-AAD. Cells beginning to die labelwith Annexin V, and as membrane integrity is lost, will also label with7-AAD. For each parthenolide derivative, the percentage of viable cellswas determined after 24 hours of culture at a 10 micromolarconcentration. Data are normalized to untreated control specimens. Thedata are in Table 1 for aminoparthenolide derivatives and Table 2 forthe salts of some aminoparthenolides.

Healthy human bone marrow cells were used in the above assay to test thecytotoxicity of parthenolide. Eighty-five percent of the normal cellssurvived 10 μM of parthenolide. All the aminoparthenolides evaluatedafforded results similar to parthenolide, i.e. the survival rate ofhealthy human bone marrow cells was over 85% at a concentration of 10μM.

TABLE 1 Aminoparthenolides and their antileukemic activity Reactants andReaction Yield Antileukemic Compound Solvent Conditions (%) activityParthenolide Sigma P0667 Not N.A. 10 μM, 84% applicable (N.A.)11S,11,13-Dihydro, 13- Parthenolide (100 mg), Refluxing 93  5 μM, 31%dimethylaminoparthenolide dimethylamine overnight 10 μM, 90% (DMAPT) (2Min methanol, 1 mL), 20 μM, 95% triethylamine(2 mL), ethanol (30 mL)11S,11,13-Dihydro, 13- Parthenolide (100 mg), Refluxing 88 10 μM, 60%diethylaminoparthenolide diethylamine overnight (DEAPT) (200 mg, 2.7mmol), triethylamine (2 mL), ethanol (30 mL) 11S,11,13-Dihydro, 13-Parthenolide (20 mg), Refluxing 39 10 μM, 20% (tert-butylamino)tert-butylamine 10 hours parthenolide (tBAPT) (0.2 mL), triethylamine(0.4 mL), ethanol (5 mL) 11S,11,13-Dihydro, 13- Parthenolide (30 mg),Refluxing 80  5 μM, 23% (pyrrolidin-1-yl) pyrrolidine (0.2 mL), 12 hours10 μM, 85% parthenolide (PyrPT) triethylamine 20 μM, 95% (0.2 mL),ethanol (5 mL) 11S,11,13-Dihydro, 13- Parthenolide (250 mg), Refluxing86 2.5 μM, 71%  (piperidin-1-yl)parthenolide piperidine (1 mL),overnight  5 μM, 91% (PipPT) triethylamine (5 mL), ethanol (100 mL)11S,11,13-Dihydro, 13- Parthenolide (100 mg), Refluxing 91  5 μM, 5%(morpholin-1- morpholine overnight 20 μM, 20% yl)parthenolide (MorPT)(0.5 mL), triethylamine (2 mL), ethanol (30 mL) 11S,11,13-Dihydro,13-(4- Parthenolide (100 mg), Refluxing 89 10 μM, 83% methylpiperidin-1-4- overnight yl)parthenolide methylpiperidine (4MePipPT) (0.5 mL),triethylamine (2 mL), ethanol (30 mL) 11S,11,13-Dihydro, 13-(4-Parthenolide (30 mg), Refluxing 74 10 μM, 7% methylpiperazin-1- 4-overnight yl)parthenolide methylpiperazine (4MePizPT) (0.2 mL),triethylamine (1 mL), ethanol (20 mL) 11S,11,13-Dihydro, 13-Parthenolide (100 mg), Refluxing 82 10 μM, 40% (homopiperidin-1-homopiperidine overnight yl)parthenolide (500 mg), (HomoPipPT)triethylamine (2 ml), ethanol (30 ml) 11S,11,13-Dihydro, 13-Parthenolide (100 mg), Refluxing 74 10 μM, 10% (heptamethyleneimin-1-heptamethyleneimin overnight yl)parthenolide (500 mg), (HeptaMePipt)triethylamine (2 mL), ethanol (30 mL) 11S,11,13-Dihydro, 13-Parthenolide (100 mg), Stirred at 93 (azetidin-1-yl)parthenolideazetidine (100 mg), room (AzePT) triethylamine temperature (2 mLl),ethanol (20 mL) 2 days 11S,11,13-Dihydro, 13- Parthenolide (100 mg),Refluxing 57 diallylaminoparthenolide diallylamine overnight (200 mg),triethylamine (2 mL), ethanol (30 mL) 11S,11,13-Dihydro, 13-Parthenolide 25 mg Room 85% 10 μM, 68% Methylbutyl amino-Methylbutylamine temperature parthenolide 20 mg with Methanol, 8 hrsstirring 11S,11,13-Dihydro, 13- Parthenolide 25 mg Room 88% 10 μM, 45%Methyl pentyl amino Methylpentylamine temperature parthenolide 20 mgMethanol, 6 hrs with stirring 11S,11,13-Dihydro, 13- Parthenolide 25 mgRoom 90% 10 μM, 2% ethylamino Ethylamine 90 mg temperature parthenolideMethanol, 5 hrs with stirring 11S,11,13-Dihydro, 13- Parthenolide 25 mg,Room 93% 10 μM, 4% methylamino parthenolide 2M Methylamine intemperature methanol (1 ml), with Methanol, 5 hrs stirring11S,11,13-Dihydro, 13- Parthenolide 25 mg Room 90% 10 μM, −6%cyclopropylamino Cyclopropylamine temperature parthenolide 20 mgMethanol, 6 hrs with stirring 11S,11,13-Dihydro, 13- Parthenolide 25 mgRoom 82% 10 μM, 7% propargylamino Propargylamine temperatureparthenolide 20 mg Methanol, 6 hrs with stirring

TABLE 2 Aminoparthenolide salts and their antileukemic activity ReactionYield Antileukemic Compound Reactants and Solvent Conditions (%)activity 11S,11,13-Dihydro, 13- 11S,11,13-Dihydro, 13- Refrigerator, 72dimethylaminoparthenolide dimethylaminoparthenolide 24 hourshydrochloride (10 mg), HCl in ether (1M, 0.03 mL) 11S,11,13-Dihydro, 13-11S,11,13-Dihydro, 13- Refrigerator, 10 10 μM, 85% (pyrrolidin-1-(pyrrolidin-1- 24 hours yl)parthenolide yl)parthenolide (5 mg),hydrochloride HCl in ether (1M, 0.015 mL) 11S,11,13-Dihydro, 13-11S,11,13-Dihydro, 13- Refrigerator, 18 10 μM, 88% (piperidin-1-(piperidin-1- 24 hours yl)parthenolide yl)parthenolide (5 mg),hydrochloride HCl in ether (1M, 0.015 mL) 11S,11,13-Dihydro, 13-11S,11,13-Dihydro, 13- Refrigerator, 38.3 10 μM, 62%(4-methylpiperidin-1- (4-methylpiperidin-1- 4 days yl)parthenolideyl)parthenolide (50 mg), hydrochloride HCl in ether (1M, 0.15 mL)11S,11,13-Dihydro, 13- 11S,11,13-Dihydro, 13- Room 55dimethylaminoparthenolide dimethylaminoparthenolide temperature maleate(30 mg), maleic acid for 1 (12 mg), ethanol (8 mL) week11S,11,13-Dihydro, 13- 11S,11,13-Dihydro, 13- Room 86dimethylaminoparthenolide dimethylaminoparthenolide temperaturemethiodide (30 mg), added for 3 days iodomethane (90 mg), methanol (6mL) 11S,11,13-Dihydro, 13- 11S,11,13-Dihydro, 13- Room 79(4-methylpiperidin-1- (4-methylpiperidin-1- temperature yl)parthenolideyl)parthenolide (70 mg), for 3 days methiodide iodomethane (200 mg),methanol (12 mL) 11H,13-(N,N- 10 μM, 12% Dimethylamino)-dehydrocostuslactone 11H,13-(N-Piperidine)- 10 μM, 14%dehydrocostuslactone 11H,13-(N-Butyl-N- 10 μM, 8% methylamino)-dehydrocostuslactone 11H,13-(N- 10 μM, 24% Propylamino)-dehydrocostuslactone 11H,13-(N-Diallyl)- 10 μM, 2% dehydrocostuslactone11H,13-(N- 10 μM, 10% Morpholine)- dehydrocostuslactone11H,13-(N-Pyrrolidine)- 10 μM, 20% dehydrocostuslactone11H,13-(N-Ethyl-N- 10 μM, 13% benzyl)- dehydrocostuslactone11H,13-(N-Methyl-N- 10 μM, 1% propylamino)- dehydrocostuslactone11H,13-(N,N- 10 μM, 5% Diethylamino)- dehydrocostuslactone11H,13-(N-Methyl-N- 10 μM, 0% pentylamino)- dehydrocostuslactone

Example 10 Analysis of Parthenolide and Dimethylaminoparthenolide(DMAPT) Using Human-Mouse Xenografts

To assess the effect of parthenolide on primary human stem cellpopulations, experiments were conducted using transplantation intoimmune deficient NOD/SCID mice. Successful engraftment of NOD/SCID bonemarrow at 6-8 weeks post-transplant has been shown to be a measure ofstem cell content for human hematopoietic cell populations (Lapidot etal., J Mol Med. 1997; 75: 664-673; Dick, Curr Opin Hematol. 1996;3:405-409). For each experiment, cryopreserved mononuclear cellspecimens from normal or AML donors were thawed, and treated in vitrowith 7.5 micromolar parthenolide for 12-18 hours. Following culture,5-10 million cells/animal were injected intravenously into sublethallyirradiated (300 Rad) NOD/SCID mice. After 6-8 weeks, animals weresacrificed and bone marrow was analyzed for the presence of human cellsusing flow cytometry as previously described (Guzman et al., Proc NatlAcad Sci USA 2002; 99: 16220-162253). Human specific antibodies for CD45were used to assess the level of total engraftment.

In three independent experiments, the level of engraftment forparthenolide-treated AML cells was dramatically reduced, which indicatesa direct effect on the AML stem cell compartment. In contrast, noreduction in engraftment was detected for parthenolide-treated normalspecimens, thus showing the parthenolide does not target normalhematopoietic stem cells. Similarly, treatment of AML cells with 7.5micromolar DMAPT also yielded a strong reduction in NOD/SCID engraftmentwhile treatment of normal cells showed no significant effects.

Example 11 MTS-PMS Assay

A 96-well U-bottomed plate (Becton Dickinson Labware, Franklin Lakes,N.J.) at a concentration of 5,000 cells per 50 microliters (mL) of mediawas incubated in 5% CO₂ at 37° C. for 24 hours. Varying compoundconcentrations in 50 mL of media were added to the media 24 hours later.Colorimetric readings were obtained using the MTS/PMS system and anELISA plate reader, after 48 hours of exposure to DMAPT. The readingsobtained for each concentration tested were from an average of eightwells. Each experiment was expressed as a percentage of the solventcontrol and completed at least three times with consistent results. Theresults presented are an average of three experiments. The hormonerefractory prostate cancer cell line CWR22Rv1 was treated withincreasing concentrations of parthenolide and DMAPT, for three hoursBoth parthenolide and DMAPT reduced cellular proliferation by 50% at 5μm in the CWR22 MTS-PMS assay. Cellular proliferation was also measuredin the MTS-PMS assay using four lung cancer cell lines treated withparthenolide and derivatives PIPT((11S,11,13-dihydro,13-(piperidin-1-yl)parthenolide), 4MEPT(11S,11,13-dihydro,13-(4-methylpiperidin-1-yl)parthenolide) and MAPT(11S,11,13-dihydro,13-dimethylaminoparthenolide). Parthenolide and itsderivatives inhibited cellular proliferation in a dose dependent mannerbetween 2 and 10 μM with 70% inhibition at 10 μM in A549, 50% in H460,40% in H-23 and 40% in H522 (FIGS. 2-5).

Example 12 Clonogenic Assay

Initially, 100 cells growing in log phase were plated per 3 ml of mediain each well of a six well plate. After 24 hrs of plating of the cellsthe test compound was added at varying concentrations. At 24 and 96hours after addition of drug, the media was changed. Hence, the cellswere only exposed to the drug for 24 hrs. When cell colonies appeared atDay 15 they were stained by Sure Stain Dye and counted. The hormonerefractory prostate cancer cell line CWR22Rv1 was treated withincreasing concentrations of parthenolide derivatives DMAPT, PIPT and4MEPT for three hours. Cellular proliferation was reduced by up to 80%at 2 μm in the clonogenic assay (FIG. 1). The breast cancer cell lineclonogenic assay with hbl-100, mdl-231 and 436 cells showed almostcomplete inhibition of proliferation with DMAPT at 2 μm concentration inthe clonogenic assay (FIGS. 6-8). Parthenolide also reducedproliferation with similar dosage ranges.

Example 13 cDNA Array Analysis

Total cellular RNA was extracted from the human monocyte cell line THP-1under three conditions 2 hours after Time 0:

-   -   1) Control was added at Time 0    -   2) Lipopolysacchride (10 nM) was added at Time plus one (1) hour    -   3) At Time 0, 10 micromoles of DMAPT was added and then at        Time+1 LPS (10 nM) was added.

RNA was extracted using RNeasy Min Kit (Qiagen, USA) according to themanufacturer's instructions. The Human Drug Targets for Inflammation andImmunomodulation Q series GE array kit (HS-048-12) was obtained fromSuperArray Bioscience Corporation (Frederick, Md.). The kit determinesexpression of 96 genes that are associated with inflammation. RNA fromrespective samples was used as a template to generate biotin labeledcDNA probes using GEArray Ampolabelling RT kit (SuperArray, BioscienceCorp., USA). The cDNA probes corresponding to the mRNA population werethen denatured and hybridization was carried out in GEHyb solution tonylon membranes spotted with gene specific fragments. Membranes werethen washed in 2×SSC, 1% SDS twice for 15 minutes each, followed by 0.1SSC, 0.5% SDS twice for 15 minutes each. Chemiluminescence was used tovisualize the expression levels of each transcript and the results werequantified with the GEArray Analyzer. The change in a given genetranscript was estimated by normalizing the signal intensities with thesignal derived from PPIA and with minimum background subtraction.

As can be seen in Table 3, transcription of 25 genes was increased afterpre-treatment with LPS. More importantly pretreatment with DMAPTprevented or blunted the increase in gene transcription induced by LPS.For example, the transcription of tumor necrosis factor (TNF), releasedin septic shock, is increased by 3 fold (298%) when treated with LPS.Pretreatment with DMAPT however prevents transcription of LPS and infact decreases its production to 2% of control. Similarly, transcriptionof cyclo-oxygenase-2, the target of classical non-steroidalanti-inflammatory agents, was increased 1.5 fold (150%). In the presenceof DMAPT, the gene expression not only prevented the increase by LPS butdecreased it to 30% (0.7) of solvent control. DMAPT therefore may act todecrease inflammation by decreasing cytokines as evidenced by decreasedgenes in human monocytes

TABLE 3 cDNA Array Analysis DMAPT Pre-treatment for LPS 2 hoursTreatment then 1 hour for Treatment 1 hour: of LPS % Change % ChangeGene of Gene of Genes CD28 antigen (Tp44) 23 0.814 CD3G antigen, gammapolypeptide (TiT3 14 0.6 complex) Colony stimulating factor 2 26 0.926(granulocyte-macrophage) Intercellular Adhesion Molecule 1 257 58Interleukin 13 93 0.64 Interleukin 1 receptor, type I 10 0.33Interleukin 1 receptor, type II 326 0.74 Nitric oxide synthase 2A(inducible) 226 48 Phosphodiesterase 4A, cAMP-specific 14 0.46Phosphodiesterase 4B, cAMP-specific 220 0.59 Phospholipase A2, group IB(pancreas) 114 0.57 Phospholipase A2, group IVC 350 0.89 PhospholipaseA2, group VII 129 0.05 Phospholipase C, gamma 1 342 0.24 Peroxisomeproliferative activated receptor, 49 0.48 gamma Platelet-activatingfactor receptor 32 0.002 Prostaglandin D2 receptor (DP) 35 0.17Prostaglandin F receptor (FP) 879 1.46 Cyclooxygenase 1 176 0.731Cyclooxygenase 2 152 0.7 Thromboxane A synthase 1 283 0.07 Tumornecrosis factor 298 0.02 (TNF superfamily, member 2) Tumor necrosisfactor (ligand) superfamily, 217 0.89 member 13b Tumor necrosis factor(ligand) superfamily, 692 23 member 5 Vascular cell adhesion molecule 1154 0.02

Example 14 Oral Bioavailability in Mice

Preliminary in vivo work was conducted to determine the bioavailabilityand toxicity of this agent in mice. As shown in FIG. 4 it wasdemonstrated that whereas 40 mg/kg of oral parthenolide provided aplasma level one hour after oral gavage of only ˜200 nM, the same doseof DMAPT provided a plasma level of ˜2500 ng/mL (ie 8 μM—average of 5mice in each group; measured by LC-MS). Given the concern from providingsuch a high plasma concentration we completed a preliminary toxicitystudy that showed the mice gained weight and survived three weeks ofdaily treatment with oral DMAPT at 40 mg/kg with no overt toxicities.

Example 15 Electrophoretic Mobility Gel Shift Assay

Each cancer cell line in exponential growth phase was treated withsolvent control or various concentrations of parthenolide derivativesdissolved in 100% ethanol for 3 hours prior to harvesting. Cells wereharvested and whole cell extracts were prepared as described previously(Nakshatri et al., Mol Cell Biol, 17: 3629-3639, 1997; Sweeney et al.,Clin Cancer Res, 10: 5501-5507, 2004). Extracts were incubated with aradiolabelled NFκB probe for 30 minutes at room temperature. Theoligonucleotide probe binds to the NFκB DNA binding site in the promoterregion of the immunoglobulin gene. Electrophoresis and autoradioragraphywere performed as described previously (Nakshatri et al., 1997) usingNFκB and SP-1 probes (Promega, Madison, Wis.). The specificity ofparthenolide and derivative inhibition of NFκB DNA binding was verifiedby the use of the SP-1 probe as a control. Identification of the NFκBsubunits binding to DNA and inhibited by DMAPT were identified by gelsupershift. Constitutive NF-κB DNA binding activity was determined inthe lung cancer cell lines A-549, H-23, H-522, and H-460. All fournon-small lung cancer cells were treated with increasing concentrationsof DMAPT for three hours, and NF-κB DNA binding was measured byelectrophoretic mobility shift assay (EMSA) as described. NF-κB DNAbinding activity was highest in A-549 cells, followed by H-23, H-522 andH-460 cells. DMAPT between 2 and 10 micromolar substantially decreasedNF-κB DNA binding activity in all lung cancer cell lines tested.

Cancer cell lines HT-1376 and UMUC-3 were treated with increasingconcentrations of DMAPT for three hours. Whole cell extracts wereprepared as described and DNA binding by NF-κB was analyzed by EMSA withNF-κB and OCT-1 (internal control) probes. DMAPT decreased NF-κB DNAbinding in a dose-dependent manner with HT-1376 and UMUC-3 cell lines(FIG. 10).

The hormone refractory prostate cancer cell line, CWR22Rv1 was treatedwith increasing concentrations of DMAPT for three hours. Whole cellextracts were prepared as described and DNA binding by NF-κB wasanalyzed by EMSA with NF-κB and SP-1 (internal control) probes. DMAPTdecreased NF-κB DNA binding in a dose dependent manner with substantialdecreases of NF-κB DNA binding at 10 μM DMAPT.

EMSA results thus showed DMAPT decreased the constitutive NF-κB DNAbinding in several cancer cell lines.

Example 16 Pretreatment of Radiation Sensitive Cell Line A549

The radiation sensitive cell line A549 was pretreated with parthenolideconcentrations ranging from 0 to 2.5 micromolar. The cells were thensubjected to ionizing radiation doses ranging from 0-6Gy and survivalfraction of the cells determined. Results demonstrated that parthenolideinduced radiation sensitivity to the cells in a dose-dependent mannerwith survival fraction at 2.5 micromolar ranging from 10% at 2Gy to lessthan 1% at 6Gy. Cells not receiving pre-treatment with parthenolide hadgreater than 50% survival fraction at the highest radiation dose of 6Gyand over 90% survival at 2Gy.

Example 17 TRAIL Induced Apoptosis Assay

MDA-MB-231 breast cancer cells (2×105 cells in 60 mm plates) weretreated first with 2 or 5 μM of DMAPT (LC-1). After two hours, TRAIL(TNF related-apoptosis-inducing-ligand, 5 ng/ml) or TRAIL-RII-activatingantibodies (10 ng/ml) were added. After 48 hours of TRAIL or TRAIL-RIIantibody treatment, cells were harvested and apoptosis was measuredusing carboxyfluorescein-FLICA assay. Briefly, both attached andfloating cells were collected by trypsinization, incubated withcarboxyfluorescein-labeled pan-caspase inhibitor FAM-VAD-FMK for 2 h at37° C. Labeled cells were rinsed twice in PBS and resuspended in 300 μlof PBS containing 0.3 μg of propidium iodide. Apoptotic cells wereidentified by FACScan analysis. Live cells do not stain (FIGS. 11 and12) Lower left quadrant). FAM-VAD-FMK stains apoptotic cells (upperleft). Apoptotic cells that have lost plasma membrane integrity arestained by both FAM-VAD-FMK and propidium iodide (upper right). Necroticcells are stained only by propidium iodide (lower right). MDA-MB-231cells are relatively resistant to TRAIL. However, they became sensitiveto TRAIL or TRAIL-RII activating antibody-induced apoptosis and atypicalapoptosis upon pre-treatment with DMAPT.

We claim:
 1. A method of inhibiting cancer cell growth comprisingcontacting said cancer cell in vitro with an effective amount of acompound of formula (I):

wherein: X₁, X₂ and X₃ are O; R₅, R₆, R₇, R₉, and R₁₀ are H, and R₄ andR₈ are methyl; and Z is —CH₂R* wherein R* is an amino acid residuebonded to the Z methylene via a nitrogen or a sulfur atom; or R* is—NR¹CO₂—R², —NR¹C(O)NR², —S—R¹, —NR¹R², or —N⁺R¹R²R¹¹Y⁻ wherein R¹ andR² are independently selected from H, CN, and optionally substitutedstraight-chained or branched aliphatic optionally containing 1 or moredouble or triple bonds; wherein optional substituents are selected fromone or more of —NH₂, —NH(C₁₋₄ aliphatic), —N(C₁₋₄ aliphatic)₂, halogen,—OH, —O—(C₁₋₄ aliphatic), —NO₂, —CN, —CO₂H, —CO₂(C₁₋₄ aliphatic),—O(halo C₁₋₄ aliphatic), or -halo(C₁₋₄ aliphatic); wherein each C₁₋₄aliphatic is optionally substituted; or R¹ and R² are independentlyselected from cycloalkyl, heterocycloalkyl, aryl or heteroaryl; andprovided that R¹ and R² are not simultaneously H; or where R* is NR¹R²,R¹ and R² optionally together with the nitrogen atom form an optionallysubstituted 5-12 membered ring, said ring optionally comprising 1 ormore heteroatoms or a group selected from —CO—, —SO—, and —SO₂—; R¹¹ isselected from H or C₁₋₄ aliphatic; and Y⁻ is selected from the groupconsisting of fluoride, chloride, bromide, iodide, sulfate, nitrate,bicarbonate, carbonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, alpha-ketoglutarate,alpha-glycerophosphate, methylsulfonate, toluenesulfonate, andbenzenesulfonate; or a pharmaceutically acceptable salt thereof.
 2. Themethod of claim 1 wherein Z is —CH₂—NR¹R².
 3. The method of claim 2wherein R¹ and R² are independently selected from hydrogen, —CN oroptionally substituted C₁-C₄ alkyl.
 4. The method of claim 3 wherein R¹and R² are independently selected from —NO₂, —CN, —CH₃, —CF₃, —CH₂CH₃,—CH₂CF₃, —CH₂Cl, —CH₂OH, —CH₂CH₂OH and —CH₂NH₂.
 5. The method of claim 2wherein R¹ and R² together with N form an optionally substituted ring.6. The method of claim 5 wherein said ring is a monocyclic, bicyclic ortricyclic alkyl or aryl ring system, said ring system optionallysubstituted and optionally comprising one or more heteroatoms or a groupselected from —CO—, —SO—, and —SO₂.
 7. The method of claim 6 wherein R¹and R² are —CH₂(CH₂)_(n)CH₂Y—; where Y is a heteroatom or a groupselected from —CO—, —SO—, and —SO₂—; n is an integer 0 to 5; andtogether with N form an optionally substituted ring, said ringoptionally fused to a cycloalkyl or aryl group to form a bicyclic ortricyclic ring system, said system optionally substituted and optionallycomprising one or more heteroatoms.
 8. The method of claim 6 wherein R₁and R₂ are —(CH₂)_(a)—Y—(CH₂)_(b)—; where Y is a heteroatom or a groupselected from —CO—, —SO—, and —SO₂—; a is an integer 0 to 5; b is aninteger 0 to 5; the sum of a and b being 0 to 5; and together with Nform an optionally substituted ring, said ring optionally fused to acycloalkyl or aryl group to form a bicyclic or tricyclic ring system,said system optionally substituted and optionally comprising one or moreheteroatoms.
 9. The method of claim 6 wherein NR₁R₂ is selected fromoptionally substituted aziridin-1-yl, azetidin-1-yl, pyrrolidin-1-yl,piperidin-1-yl, homopiperidyn-1-yl and heptamethyleneimin-1-yl.
 10. Themethod of claim 1 wherein the compound of formula (I) is selected from:11S,11,13-Dihydro,13-dimethylaminoparthenolide;11S,11,13-Dihydro,13-diethylaminoparthenolide;11S,11,13-Dihydro,13-(tert-butylamino)parthenolide;11S,11,13-Dihydro,13-(pyrrolidin-1-yl)parthenolide;11S,11,13-Dihydro,13-(piperidin-1-yl)parthenolide;11S,11,13-Dihydro,13-(morpholin-1-yl)parthenolide;11S,11,13-Dihydro,13-(4-methylpiperidin-1-yl)parthenolide;11S,11,13-Dihydro,13-(4-methylpiperazin-1-yl)parthenolide;11S,11,13-Dihydro,13-(homopiperidin-1-yl)parthenolide;11S,11,13-Dihydro, 13-(heptamethyleneimin-1-yl)parthenolide;11S,11,13-Dihydro,13-(azetidin-1-yl)parthenolide;11S,11,13-Dihydro,13-methylbutyl aminoparthenolide;11S,11,13-Dihydro,13-methyl pentyl aminoparthenolide;11S,11,13-Dihydro,13-ethylaminoparthenolide;11S,11,13-Dihydro,13-methylaminoparthenolide;11S,11,13-Dihydro,13-cyclopropylaminoparthenolide;11S,11,13-Dihydro,13-propargylaminoparthenolide;11S,11,13-Dihydro,13-(N-benzyl-N-ethylamine)parthenolide;11S,11,13-Dihydro,13-(N-prolyl)parthenolide;11S,11,13-Dihydro,13-(S-thiophenolyl)parthenolide;11S,11,13-Dihydro,13-(N,N-diethanolamine)parthenolide;11S,11,13-Dihydro,13-(thiomorpholin-4-yl)parthenolide;11S,11,13-Dihydro,13-(4-hydroxypiperidin-1-yl)parthenolide;11S,11,13-Dihydro,13-(1-methylhomopiperizin-4-yl)parthenolide;11S,11,13-Dihydro,13-(S-mercaptoacetyl)parthenolide;11S,11,13-Dihydro,13-(4-(2′-hydroxyethyl)piperidin-1-yl)parthenolide;11S,11,13-Dihydro,13-(piperazin-1-yl-4-carboxaldehyde)parthenolide;11S,11,13-Dihydro,13-(4-benzylpiperidin-1-yl)parthenolide;11S,11,13-Dihydro,13-(piperidin-1-yl-4-carboxylic acid)parthenolide;11S,11,13-Dihydro,13-(azetidin-1-yl-3-carboxylic acid)parthenolide;11S,11,13-Dihydro,13-(S-cysteinyl)parthenolide;11S,11,13-Dihydro,13-(4-(piperidin-1′-yl)piperidin-1-yl))parthenolide;11S,11,13-Dihydro,13-diallylaminoparthenolide; or a pharmaceuticallyacceptable salt thereof.
 11. The method of claim 10 wherein the compoundof formula (I) is 11S,11,13-Dihydro,13-dimethylaminoparthenolide; or apharmaceutically acceptable salt thereof.
 12. The method of claim 11wherein the compound of formula (I) is11S,11,13-Dihydro,13-dimethylaminoparthenolide hydrochloride; or11S,11,13-Dihydro,13-dimethylaminoparthenolide maleate.
 13. The methodof claim 1 wherein the compound of formula (I) is selected from:11S,11,13-Dihydro,13-(pyrrolidin-1-yl)parthenolide hydrochloride;11S,11,13-Dihydro,13-(piperidin-1-yl)parthenolide hydrochloride;11S,11,13-Dihydro,13-(4-methylpiperidin-1-yl)parthenolide hydrochloride;11S,11,13-Dihydro,13-dimethylaminoparthenolide methiodide; or11S,11,13-Dihydro,13-(4-methylpiperidin-1-yl)parthenolide methiodide.14. The method of claim 1 wherein Z is —CH₂S—R¹; wherein R¹ is selectedfrom optionally substituted straight-chained or branched aliphaticwherein optional substituents are selected from one or more of —NH₂,—N(C₁₋₄ aliphatic)₂, halogen, —OH, —O—(C₁₋₄ aliphatic), —NO₂, —CN,—CO₂H, —CO₂(C₁₋₄ aliphatic), —O-(halo C₁₋₄ aliphatic), or -(halo C₁₋₄aliphatic); wherein each C₁₋₄ aliphatic is optionally substituted;cycloalkyl, heterocycloalkyl, aryl or heteroaryl.
 15. The method ofclaim 1 wherein Z is —CH₂S—R¹; wherein R¹ is selected from optionallysubstituted straight-chained or branched aliphatic wherein optionalsubstituents are selected from one or more of —NH₂, —N(C₁₋₄ aliphatic)₂,halogen, —OH, —O—(C₁₋₄ aliphatic), NO₂, —CN, —CO₂H, —CO₂(C₁₋₄aliphatic), —O-(halo C₁₋₄ aliphatic), or -(halo C₁₋₄ aliphatic); whereineach C₁₋₄ aliphatic is optionally substituted; cycloalkyl,heterocycloalkyl, aryl or heteroaryl.